Hypertonic pharmaceutical compositions containing an anti-platinum chemoprotectant agent

ABSTRACT

Hypertonic pharmaceutical compositions are disclosed. The hypertonic pharmaceutical compositions contain an anti-platinum chemoprotectant agent and a gelling agent. Also disclosed are methods of medical use of the hypertonic pharmaceutical compositions.

FIELD OF THE INVENTION

The present invention provides pharmaceutical compositions containing ananti-platinum chemoprotectant agent, methods of using the same, andmethods of their preparation.

BACKGROUND

Platinum-based antineoplastic agents (e.g., cisplatin) arechemotherapeutic agents widely used to treat cancers and tumors. Theseagents are toxic and are known to induce hearing loss both in human andanimal models. Thus, patients undergoing chemotherapy withplatinum-based antineoplastic agents can suffer from hearing loss. Thereis a need for otoprotective compositions and methods to prevent ormitigate hearing loss associated with chemotherapeutic regimensincluding platinum-based antineoplastic agents.

SUMMARY OF THE INVENTION

In general, the invention provides pharmaceutical compositions (e.g.,hypertonic pharmaceutical compositions) and methods of their use forpreventing or mitigating platinum-induced ototoxicity.

In one aspect, the invention provides a pharmaceutical composition(e.g., a hypertonic pharmaceutical composition) containing ananti-platinum chemoprotectant agent and a gelling agent.

In some embodiments, the calculated osmolarity of the composition is atleast 400 mOsm/L (e.g., at least 500 mOsm/L, at least 600 mOsm/L, atleast 700 mOsm/L, at least 800 mOsm/L, at least 900 mOsm/L, at least1,000 mOsm/L, at least 1,500 mOsm/L, at least 2,000 mOsm/L, at least2,500 mOsm/L, or at least 3,000 mOsm/L). In certain embodiments, thecalculated osmolarity of the composition is 5,000 mOsm/L or less (e.g.,4,500 mOsm/L or less, 4,000 mOsm/L or less, 3,000 mOsm/L or less, 2,000mOsm/L or less, 1,800 mOsm/L or less, 1,500 mOsm/L or less, 1,200 mOsm/Lor less, or 1,000 mOsm/L or less). In some embodiments, the calculatedosmolarity of the composition is 1,500-4,500 mOsm/L. In otherembodiments, the calculated osmolarity of the composition is 3,000-4,500mOsm/L. In particular embodiments, the measured osmolality of thecomposition is at least 0.3 Osm/kg (e.g., at least 0.5 Osm/kg, at least0.6 Osm/kg, at least 0.7 Osm/kg, at least 0.8 Osm/kg, at least 0.9Osm/kg, at least 1.0 Osm/kg, at least 1.2 Osm/kg, at least 1.4 Osm/kg,or at least 1.8 Osm/kg). In further embodiments, the measured osmolalityof the composition is 2.5 Osm/kg or less (e.g., 2.1 Osm/kg or less). Inyet further embodiments, the measured osmolality of the composition is0.3-2.5 Osm/kg (e.g., 0.5-2.5 Osm/kg, 0.6-2.5 Osm/kg, 0.7-2.5 Osm/kg,0.8-2.5 Osm/kg, 0.9-2.5 Osm/kg, 1.0-2.5 Osm/kg, 1.2-2.5 Osm/kg, 1.4-2.5Osm/kg, 1.8-2.5 Osm/kg, 0.5-2.1 Osm/kg, 0.6-2.1 Osm/kg, 0.7-2.1 Osm/kg,0.8-2.1 Osm/kg, 0.9-2.1 Osm/kg, 1.0-2.1 Osm/kg, 1.2-2.1 Osm/kg, 1.4-2.1Osm/kg, or 1.8-2.1 Osm/kg).

In particular embodiments, the anti-platinum chemoprotectant agent is analkaline or ammonium thiosulfate salt or solvate thereof, an alkalinediethyldithiocarbamate salt, amifostine, methionine, N-acetylcysteine,cysteine, 2-aminoethanethiol, glutathione (GSH) or a C₁-C₆ alkyl esterthereof, lysine, histidine, arginine, ethylene diamine tetraacetic acid,dimercaprol, dimercaptosuccinic acid, dimercapto-propane sulfonate salt,penicillamine, α-lipoic acid, or fursultiamine, or a salt thereof. Infurther embodiments, the anti-platinum chemoprotectant agent is analkaline or ammonium thiosulfate salt or a solvate thereof (e.g., sodiumthiosulfate or a solvate thereof).

In yet further embodiments, the gelling agent is hyaluronan, apolyoxyethylene-polyoxypropylene block copolymer (e.g., a poloxamer),poly(lactic-co-glycolic) acid, polylactic acid, polycaprolactone,alginic acid or a salt thereof, polyethylene glycol, a cellulose, acellulose ether (e.g., methylcellulose, carboxymethylcellulose,ethylcellulose, hydroxyethylcellulose, methyl hydroxyethylcellulose,hydroxypropyl methylcellulose, or hydroxypropylcellulose), a carbomer(e.g., Carbopol®), agar-agar, gelatin, glucomannan, galactomannan (e.g.,guar gum, locust bean gum, or tara gum), xanthan gum, chitosan, pectin,starch, tragacanth, carrageenan, polyvinylpyrrolidone, polyvinylalcohol, paraffin, petrolatum, silicates, fibroin, or a combinationthereof. In still further embodiments, the gelling agent is hyaluronan.In other embodiments, the gelling agent is a combination of hyaluronanand methylcellulose. In yet other embodiments, the gelling agent is apolyoxyethylene-polyoxypropylene block copolymer (e.g., poloxamer (e.g.,poloxamer 407 or a combination of poloxamer 407 and poloxamer 188)). Instill other embodiments, the gelling agent is fibroin. In someembodiments, the pharmaceutical composition contains at least about 0.5%(w/v) (e.g., at least 0.8% (w/v), at least 1% (w/v), or at least 2%(w/v)) of the gelling agent relative to the liquid solvent. In certainembodiments, the pharmaceutical composition contains about 20% (w/v) orless (e.g., 15% (w/v) or less) of the gelling agent relative to theliquid solvent. In further embodiments, the pharmaceutical compositioncontains about 2% (w/v) or less of the gelling agent relative to theliquid solvent. In yet further embodiments, the pharmaceuticalcomposition contains about 0.8% (w/v) of the gelling agent relative tothe liquid solvent. In still further embodiments, the pharmaceuticalcomposition contains about 1% (w/v) of the gelling agent relative to theliquid solvent.

In particular embodiments, the gelling agent is cross-linked (e.g.,ionically cross-linked or covalently cross-linked). In otherembodiments, the gelling agent is not cross-linked. In particularembodiments, the pharmaceutical compositions further include apharmaceutically acceptable liquid solvent (e.g., water).

In yet other embodiments, the concentration of the anti-platinumchemoprotectant agent is at least about 0.05M (e.g., at least about0.1M, at least about 0.2M, at least about 0.3M, at least about 0.4M, atleast about 0.5M, or at least about 1M). In still other embodiments, theconcentration of the anti-platinum chemoprotectant agent is about 2.5Mor less (e.g., 2.0M or less, 1.5M or less, 1.0M or less, 0.5M or less,about 0.3M or less, or about 0.2M or less). In certain preferredembodiments, the concentration of the anti-platinum chemoprotectantagent is about 0.5M to about 1.5M. In more preferred embodiments, theconcentration of the anti-platinum chemoprotectant agent is about 1.0Mto about 1.5M.

In some embodiments, the pharmaceutical composition has a pH of 5.0 to8.5. In certain embodiments, the pharmaceutical composition has a pH of6.0 to 8.5. In particular embodiments, the pharmaceutical compositionhas a pH of 6.5 to 8.5.

In further embodiments, the pharmaceutical composition is apharmaceutical dosage form.

In another aspect, the invention provides a method of preventing ormitigating platinum-induced ototoxicity in a subject by administering tothe round window of the subject an effective amount of thepharmaceutical composition (e.g., pharmaceutical dosage form) of theinvention. In certain embodiments, the platinum-induced ototoxicity isin the subject receiving a platinum-based antineoplastic agent (e.g.,cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate,phenanthriplatin, picoplatin, or satraplatin).

In some embodiments, the pharmaceutical composition is administeredintratympanically or transtympanically. In particular embodiments, thepharmaceutical composition is administered before or after (e.g., withinabout 24, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hours) theadministration of a platinum-based antineoplastic agent. In certainembodiments, the pharmaceutical composition is administered at the sametime as the administration of a platinum-based antineoplastic agent. Infurther embodiments, the pharmaceutical composition is administered by aroute different from the platinum-based antineoplastic agent (e.g., theplatinum-based antineoplastic agent is administered parenterally (e.g.,intratumorally, intramuscularly, or intravenously)). In yet furtherembodiments, at least 50 μL (preferably, at least 100 μL; morepreferably, at least 200 μL) of the pharmaceutical composition areadministered to the round window of the subject. In still furtherembodiments, 1 mL or less of the pharmaceutical composition areadministered to the round window of the subject. In other embodiments,the subject is human.

In yet another aspect, the invention provides a method of preparing apharmaceutical composition (e.g., a pharmaceutical dosage form) of theinvention by (i) providing the anti-platinum chemoprotectant agent andthe gelling agent, and (ii) mixing the anti-platinum chemoprotectantagent and the gelling agent with the liquid solvent to produce thehypertonic pharmaceutical composition.

In some embodiments, the anti-platinum chemoprotectant agent and thegelling agent are provided as a mixture. In certain embodiments, theanti-platinum chemoprotectant agent and the gelling agent are providedseparately, and step (ii) comprises mixing the liquid solvent first withthe gelling agent to produce an intermediate mixture and thereaftermixing the intermediate mixture with the anti-platinum chemoprotectantagent. In particular embodiments, the anti-platinum chemoprotectantagent and the gelling agent are provided separately, and step (ii)comprises mixing the liquid solvent first with the anti-platinumchemoprotectant agent to produce an intermediate mixture and thereaftermixing the intermediate mixture with the gelling agent. In furtherembodiments, the anti-platinum chemoprotectant agent and the gellingagent are provided separately, and step (ii) comprises mixing a portionof the liquid solvent with the anti-platinum chemoprotectant agent toproduce a first mixture, mixing another portion of the liquid solventwith the gelling agent to produce a second mixture, and combining thefirst and second mixtures.

Definitions

The term “about,” as used herein, represents a value that is in therange of ±10% of the value that follows the term “about.”

The term “alkaline salt,” as used herein, represents a sodium orpotassium salt of a compound. Alkaline salts may be monobasic or, if thenumber of acidic moieties (e.g., —COOH, —SO₃H, or —P(O)(OH)_(n)moieties) permits, dibasic or tribasic.

The term “ammonium salt,” as used herein, represents an NH₄+ salt of acompound. Ammonium salts may be monobasic or, if the number of acidicmoieties (e.g., —COOH, —SO₃H, or —P(O)(OH)_(n) moieties) permits,dibasic or tribasic.

The term “anti-platinum chemoprotectant agent,” as used herein, refersto a compound that deactivates platinum-based antineoplastic agents.Without wishing to be bound by theory, an anti-platinum chemoprotectantagent may coordinate to the platinum center of a platinum-basedantineoplastic agent, thereby reducing the amount of active platinumcenters available for reaction with peptides and/or nucleotides presentin a subject. Non-limiting examples of anti-platinum chemoprotectantagents include an alkaline or ammonium thiosulfate salt (e.g., sodiumthiosulfate, potassium thiosulfate, or ammonium thiosulfate) or asolvate thereof (e.g., sodium thiosulfate pentahydrate), an alkalinediethyldithiocarbamate salt, amifostine, methionine, N-acetylcysteine,cysteine, 2-aminoethanethiol, glutathione (GSH) or a C₁-C₆ alkyl esterthereof (e.g., glutathione ethyl ester: γ-Glu-Cys-Gly-OEt) or a saltthereof, lysine or a salt thereof, histidine or a salt thereof, arginineor a salt thereof, ethylene diamine tetraacetic acid or a salt thereof(e.g., an alkaline salt), dimercaprol, dimercaptosuccinic acid or a saltthereof (e.g., an alkaline salt), dimercapto-propane sulfonate salt(e.g., alkaline salt or ammonium salt), penicillamine, α-lipoic acid ora salt thereof (e.g., an alkaline or ammonium salt), or fursultiamine ora salt thereof. The salts of anti-platinum chemoprotectant agents arepharmaceutically acceptable salts.

The term “gelling agent,” as used herein, refers to pharmaceuticallyacceptable excipient known in the art to produce a gel upon mixing witha solvent (e.g., an aqueous solvent). Non-limiting examples of gellingagents include hyaluronan, a polyoxyethylene-polyoxypropylene blockcopolymer (e.g., a poloxamer), poly(lactic-co-glycolic) acid, polylacticacid, polycaprolactone, alginic acid or a salt thereof, polyethyleneglycol, a cellulose, a cellulose ether, a carbomer (e.g., Carbopol®),agar-agar, gelatin, glucomannan, galactomannan (e.g., guar gum, locustbean gum, or tara gum), xanthan gum, chitosan, pectin, starch,tragacanth, carrageenan, polyvinylpyrrolidone, polyvinyl alcohol,paraffin, petrolatum, silicates, fibroin, and combinations thereof.

The term “hypertonic,” as used herein in reference to pharmaceuticalcompositions, represents a pharmaceutical composition having acalculated osmolarity of 300 mOsm/L to 7,000 mOsm/L (e.g., 300 mOsm/L to2,500 mOsm/L), which corresponds to 300 mmol to 7,000 mmol (e.g., 300mOsm/L to 2,500 mmol) of ions and/or neutral molecules produced bydissolution of platinum-deactivating agent and any ionic, non-polymericexcipients in 1 L of solvent having calculated osmolarity of 0 mOsm/L.For the purpose of the present disclosure, the calculated osmolaritydoes not include ions and/or neutral molecules produced from polymericexcipients (e.g., from a gelling agent). For the purpose of thisdisclosure, polymeric excipients (e.g., a gelling agents) are deemed asnot contributing to the calculated osmolarity of the compositionsdisclosed herein.

The term “intratympanic,” as used herein in reference to a route ofadministration, means delivery to the round window by injection orinfusion through an ear canal with a temporarily removed or liftedtympanic membrane or through a port created through an auditory bullainto the middle ear of a subject.

The term “pharmaceutical composition,” as used herein, represents acomposition formulated with a pharmaceutically acceptable excipient, andmanufactured or sold with the approval of a governmental regulatoryagency as part of a therapeutic regimen for the treatment of disease ina mammal.

The term “pharmaceutical dosage form,” as used herein, represents thosepharmaceutical compositions intended for administration to a subject asis without further modification (e.g., without dilution with, suspensionin, or dissolution in a liquid solvent).

The term “pharmaceutically acceptable excipient,” as used herein, refersto any ingredient other than the anti-platinum chemoprotectant agentsand gelling agents described herein (e.g., a vehicle capable ofsuspending or dissolving the active compound) and having the propertiesof being substantially non-toxic and substantially non-inflammatory in apatient. Excipients may include, e.g., antioxidants, disintegrants, dyes(colors), emollients, emulsifiers, fillers (diluents), flavors,fragrances, preservatives, printing inks, sorbents, suspending ordispersing agents, sweeteners, liquid solvents, and buffering agents.

The term “pharmaceutically acceptable salt,” as use herein, representsthose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and animalswithout undue toxicity, irritation, allergic response and the like andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example,pharmaceutically acceptable salts are described in: Berge et al., J.Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts:Properties, Selection, and Use, (Eds. P. H. Stahl and C. G. Wermuth),Wiley-VCH, 2008. The salts can be prepared in situ during the finalisolation and purification of the compounds described herein orseparately by reacting the free base group with a suitable organic acid.Representative acid addition salts include acetate, adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like.

The term “pharmaceutically acceptable solvate” as used herein means acompound as described herein wherein molecules of a suitable solvent areincorporated in the crystal lattice. A suitable solvent isphysiologically tolerable at the dosage administered. For example,solvates may be prepared by crystallization, recrystallization, orprecipitation from a solution that includes organic solvents, water, ora mixture thereof. Examples of suitable solvents are ethanol, water (forexample, mono-, di-, tri-, tetra-, and penta-hydrates),N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC),1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When the solvate is water-based, thesolvate is referred to as a hydrate.

The term “platinum-based antineoplastic agent,” as used herein,represents a coordination compound of Pt(II) or Pt(IV). Platinum-basedantineoplastic agents are known in the art as platins. Typically,platinum-based antineoplastic agents include at least two coordinationsites at the platinum center that are occupied by nitrogenous spectatorligand(s). The nitrogenous spectator ligands are monodentate orbidentate ligands, in which the donor atom is an sp³- or sp²-hybridizednitrogen atom within the ligand. Non-limiting examples of nitrogenousspectator ligands are ammonia, 1,2-cyclohexanediamine, a picoline,phenanthren, or 1,6-hexanediamine. Non-limiting examples ofplatinum-based antineoplastic agents include cisplatin, carboplatin,oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin,picoplatin, and satraplatin.

The term “subject,” as used herein, refers to an animal (e.g., a mammal,e.g., a human). A subject to be treated according to the methodsdescribed herein may be one who is being treated with a therapeuticregimen including a platinum-based antineoplastic agent (e.g., for thetreatment of a benign tumor, malignant tumor, or cancer). The patientmay have been diagnosed with a benign tumor, malignant tumor, or cancerby any method or technique known in the art. One skilled in the art willunderstand that a subject to be treated according to the invention mayhave been subjected to standard tests or may have been identified,without examination, as one at high risk due to receiving a therapeuticregimen including a platinum-based antineoplastic agent.

The term “substantially neutral,” used herein, refers to a pH level of5.5 to about 8.5, as measured at 20° C.

The term “tonicity agent,” as used herein, refers to a class ofpharmaceutically acceptable excipients that are used to controlosmolarity of pharmaceutical compositions. Non-limiting examples of atonicity agent include substantially neutral buffering agents (e.g.,phosphate buffered saline, tris buffer, or artificial perilymph),dextrose, mannitol, glycerin, potassium chloride, and sodium chloride(e.g., as a hypertonic, isotonic, or hypotonic saline). Artificialperilymph is an aqueous solution containing NaCl (120-130 mM), KCl (3.5mM), CaCl₂) (1.3-1.5 mM), MgCl₂ (1.2 mM), glucose (5.0-11 mM), andbuffering agents (e.g., NaHCO₃ (25 mM) and NaH₂PO₄ (0.75 mM), or HEPES(20 mM) and NaOH (adjusted to pH of about 7.5)).

The term “transtympanic,” as used herein in reference to a route ofadministration, means delivery to the round window by injection orinfusion across tympanic membrane. A transtympanic injection may beperformed directly through the tympanic membrane or through a tubeembedded in the tympanic membrane (e.g., through a tympanostomy tube orgrommet).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a chart showing the changes in thiosulfate concentrationsover time in plasma, perilymph, and cerebrospinal fluid in guinea pigsadministered a gel containing 0.1M sodium thiosulfate and 20% (w/v)poloxamer 407.

FIG. 1B is a chart showing the changes in thiosulfate concentrationsover time in plasma, perilymph, and cerebrospinal fluid in guinea pigsadministered a gel containing 0.5M sodium thiosulfate and 1% (w/v)hyaluronan.

FIG. 2A is a chart showing the changes in thiosulfate concentrationsover time in plasma, perilymph, and cerebrospinal fluid in guinea pigsadministered a gel containing 0.1M sodium thiosulfate and 2% (w/v)hyaluronan.

FIG. 2B is a chart showing the changes in thiosulfate concentrationsover time in plasma, perilymph, and cerebrospinal fluid in guinea pigsadministered a gel containing 0.5M sodium thiosulfate and 2% (w/v)hyaluronan.

FIG. 3A is a chart showing the threshold sound pressure levels at 4, 24,and 32 kHz measured across five cohorts of guinea pigs (n=27 animals)during auditory brainstem response tests. Seven days prior to theauditory brainstem response tests, all guinea pigs were injectedintraperitoneally with a bolus of cisplatin. The baseline thresholdswere from historic auditory brainstem response tests on cisplatin-naïveguinea pigs (n=100 ears). The baseline thresholds are shown as a shadedarea curve.

FIG. 3B is a chart showing the threshold sound pressure levels at 4, 24,and 32 kHz measured during auditory brainstem response tests for theguinea pigs deemed to have hearing loss (n=18 animals). Seven days priorto the auditory brainstem response tests, all guinea pigs were injectedintraperitoneally with a bolus of cisplatin. The baseline thresholdswere from historic auditory brainstem response tests on cisplatin-naïveguinea pigs (n=100 ears). The baseline thresholds are shown as a shadedarea curve.

FIG. 4A is a chart showing the average threshold sound pressure levelsat 4, 24, and 32 kHz measured during auditory brainstem response testsfor the guinea pigs deemed to have hearing loss (n=18 animals). Sevendays prior to the auditory brainstem response tests, all guinea pigswere injected intraperitoneally with a bolus of cisplatin. The baselinethresholds were from historic auditory brainstem response tests oncisplatin-naïve guinea pigs (n=100 ears). The baseline thresholds areshown as a shaded area curve.

FIG. 4B is a chart showing the average threshold sound pressure levelsat 4, 24, and 32 kHz measured during auditory brainstem response (ABR)tests for the guinea pigs administered vehicle or sodium thiosulfate toone ear each followed by a cisplatin challenge. The baseline thresholdswere from historic auditory brainstem response tests on cisplatin-naïveguinea pigs (n=100 ears). The baseline thresholds are shown as a shadedarea curve.

FIG. 5 is a figure illustrating the cisplatin challenge test followingadministration of vehicle or sodium thiosulfate to one ear of a guineapig.

FIG. 6 is a chart showing the average threshold sound pressure levels at4, 24, and 32 kHz measured during auditory brainstem response (ABR)tests for the guinea pigs administered vehicle or sodium thiosulfate(0.1M, 0.5M, or 1M sodium thiosulfate gel) to one ear each followed by acisplatin challenge. The baseline thresholds were from historic auditorybrainstem response tests on cisplatin-naïve guinea pigs (n=100 ears).The baseline thresholds are shown as a shaded area curve.

DETAILED DESCRIPTION

In general, the invention provides pharmaceutical compositionscontaining anti-platinum chemoprotectant agents. The pharmaceuticalcompositions of the invention may be used in the treatment ofplatinum-induced ototoxicity in a subject receiving a platinum-basedantineoplastic agent (e.g., a subject having a tumor or cancer).Non-limiting examples of the platinum-based antineoplastic agentsinclude cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatintetranitrate, phenanthriplatin, picoplatin, and satraplatin.

The pharmaceutical compositions of the invention may prevent or mitigatehearing loss in a subject receiving a platinum-based antineoplasticagent, as measured by at least 50% (e.g., at least 60%, at least 70%, orat least 80%) reduction in the sound pressure level threshold elevationin the subject at a frequency 8 kHz or higher (e.g., between 8 kHz and20 kHz) relative to a reference subject that receives the sameplatinum-based antineoplastic agent regimen but does not receive theanti-platinum chemoprotectant agent.

The pharmaceutical compositions of the invention are hypertonic. Withoutwishing to be bound by theory, the higher tonicity of the pharmaceuticalcompositions of the invention is believed to improve the bioavailabilityof anti-platinum chemoprotectant agents at the round window of asubject, relative to compositions with lower tonicity (e.g., hypotonicor isotonic). The bioavailability is typically measured as AUC_(inf) foran anti-platinum chemoprotectant agent following its administration toan animal (e.g., a mammal). The calculated osmolarity of thepharmaceutical composition of the invention (e.g., pharmaceutical dosageform) may be, e.g., at least 400 mOsm/L (e.g., at least 500 mOsm/L, atleast 600 mOsm/L, at least 700 mOsm/L, at least 800 mOsm/L, at least 900mOsm/L, at least 1,000 mOsm/L, at least 1,500 mOsm/L, at least 2,000mOsm/L, at least 2,500 mOsm/L, or at least 3,000 mOsm/L), and/or 5,000mOsm/L or less (e.g., 4,000 mOsm/L or less, 3,000 mOsm/L or less, 2,000mOsm/L or less, 1,900 mOsm/L or less, 1,800 mOsm/L or less, 1,700 mOsm/Lor less, 1,600 mOsm/L or less, or 1,500 mOsm/L or less). The calculatedosmolarity of the pharmaceutical composition of the invention (e.g.,pharmaceutical dosage form) may be, e.g., 1,500-4,500 mOsm/L. Thecalculated osmolarity of the pharmaceutical composition of the invention(e.g., pharmaceutical dosage forms) may be, e.g., 3,000-4,500 mOsm/L.The measured osmolality of the pharmaceutical composition of theinvention (e.g., pharmaceutical dosage form) may be, e.g., at least 0.3Osm/kg (e.g., at least 0.5 Osm/kg, at least 0.6 Osm/kg, at least 0.7Osm/kg, at least 0.8 Osm/kg, at least 0.9 Osm/kg, at least 1.0 Osm/kg,at least 1.2 Osm/kg, at least 1.4 Osm/kg, or at least 1.8 Osm/kg). Themeasured osmolality of the pharmaceutical composition of the invention(e.g., pharmaceutical dosage form) may be, e.g., 2.5 Osm/kg or less(e.g., 2.1 Osm/kg or less). The measured osmolality of thepharmaceutical composition of the invention (e.g., pharmaceutical dosageform) may be, e.g., 0.3-2.5 Osm/kg (e.g., 0.5-2.5 Osm/kg, 0.6-2.5Osm/kg, 0.7-2.5 Osm/kg, 0.8-2.5 Osm/kg, 0.9-2.5 Osm/kg, 1.0-2.5 Osm/kg,1.2-2.5 Osm/kg, 1.4-2.5 Osm/kg, 1.8-2.5 Osm/kg, 0.5-2.1 Osm/kg, 0.6-2.1Osm/kg, 0.7-2.1 Osm/kg, 0.8-2.1 Osm/kg, 0.9-2.1 Osm/kg, 1.0-2.1 Osm/kg,1.2-2.1 Osm/kg, 1.4-2.1 Osm/kg, or 1.8-2.1 Osm/kg). “Calculatedosmolarity” refers to the number of mmoles of ions and/or neutralmolecules produced by dissolution of one or more compounds in 1 L of DIor distilled water; calculated osmolarity does not include ions and/orneutral molecules produced from polymeric excipients (e.g., from agelling agent). “Measured osmolality” refers to the osmolality of acomposition, as measured using an osmometer (typically, a membraneosmometer).

An anti-platinum chemoprotectant agent may be, e.g., the sole compoundcontributing to osmolarity of a pharmaceutical composition of theinvention. Alternatively, higher osmolarities than those afforded by thedesired concentration of an anti-platinum chemoprotectant agent may beachieved, e.g., through the use of tonicity agents. A tonicity agent maybe present in a hypertonic, isotonic, or hypotonic excipient (e.g., ahypotonic liquid solvent). Non-limiting examples of tonicity agentsinclude substantially neutral buffering agents (e.g., phosphate bufferedsaline, tris buffer, or artificial perilymph), dextrose, mannitol,glycerin, potassium chloride, and sodium chloride (e.g., as ahypertonic, isotonic, or hypotonic saline).

Anti-Platinum Chemoprotectant Agents

Pharmaceutical compositions of the invention contain an anti-platinumchemoprotectant agent. Without wishing to be bound by theory,anti-platinum chemoprotectant agents are believed to reduce or eliminatethe toxicity of platin-based antineoplastic agents by competitivelyligating and substantially coordinatively saturating the platinumcenters present in the platinum-based antineoplastic agents. Theconcentration of an anti-platinum chemoprotectant agent in apharmaceutical composition (e.g., a pharmaceutical dosage form) of theinvention may be, e.g., at least about 0.05M (e.g., at least about 0.1M,at least about 0.2M, at least about 0.3M, at least about 0.4M, at leastabout 0.5M, or at least about 1M). The concentration of an anti-platinumchemoprotectant agent in a pharmaceutical composition (e.g., apharmaceutical dosage form) of the invention may be, e.g., about 2.5M orless (e.g., 2.0M or less, 1.5M or less, 1.0M or less, 0.5M or less,about 0.3M or less, or about 0.2M or less). Non-limiting examples of theconcentrations of an anti-platinum chemoprotectant agent in apharmaceutical composition (e.g., a pharmaceutical dosage form) of theinvention may be, e.g., about 0.05M to about 1.5M, about 0.05M to about0.5M, about 0.05M to about 0.2M, about 0.05M to about 0.1M, about 0.1Mto about 1.5M, about 0.1M to about 0.5M, about 0.1M to about 0.2M, about0.2M to about 1.5M, about 0.2M to about 0.5M, about 0.5M to about 1.5M,0.05M to about 1.0M, about 0.05M to about 0.5M, about 0.05M to about0.2M, about 0.05M to about 0.1M, about 0.1M to about 1.0M, about 0.1M toabout 0.5M, about 0.1M to about 0.2M, about 0.2M to about 1.0M, about0.2M to about 0.5M, or about 0.5M to about 1.0M, or about 1.0M to about1.5M. Preferably, the concentration of an anti-platinum chemoprotectantagent in a pharmaceutical composition (e.g., a pharmaceutical dosageform) of the invention is about 1.0M to about 1.5M.

Anti-platinum chemoprotectant agents are known in the art. Non-limitingexamples of anti-platinum chemoprotectant agents include an alkaline orammonium thiosulfate salt (e.g., sodium thiosulfate, potassiumthiosulfate, or ammonium thiosulfate) or a solvate thereof (e.g., sodiumthiosulfate pentahydrate), an alkaline diethyldithiocarbamate salt,amifostine, methionine, N-acetylcysteine, cysteine, 2-aminoethanethiol,glutathione (GSH) or a C₁-C₆ alkyl ester thereof (e.g., glutathioneethyl ester: γ-Glu-Cys-Gly-OEt) or a salt thereof, lysine or a saltthereof, histidine or a salt thereof, arginine or a salt thereof,ethylene diamine tetraacetic acid or a salt thereof (e.g., an alkalinesalt), dimercaprol, dimercaptosuccinic acid or a salt thereof (e.g., analkaline salt), dimercapto-propane sulfonate salt (e.g., alkaline saltor ammonium salt), penicillamine, α-lipoic acid or a salt thereof (e.g.,an alkaline or ammonium salt), or fursultiamine or a salt thereof.Preferably, the anti-platinum chemoprotectant agent is an alkaline orammonium thiosulfate salt. More preferably, the anti-platinumchemoprotectant agent is sodium thiosulfate.

Gelling Agents

Pharmaceutical compositions of the invention include a gelling agent.Gelling agents may be used to increase the viscosity of thepharmaceutical composition, thereby improving the retention of thepharmaceutical composition at the targeted site. Pharmaceuticalcompositions (e.g., pharmaceutical dosage forms) of the invention maycontain, e.g., about 0.1% to about 25% (w/v) (e.g., about 0.1% to about20% (w/v), about 0.1% to about 10% (w/v), about 0.1% to about 2% (w/v),about 0.5% to about 25% (w/v), about 0.5% to about 20% (w/v), about 0.5%to about 10% (w/v), about 0.5% to about 2% (w/v), about 1% to about 20%(w/v), about 1% to about 10% (w/v), about 1% to about 2% (w/v), about 5%to about 20% (w/v), about 5% to about 10% (w/v), or about 7% to about10% (w/v)) of a gelling agent relative to solvent. Preferably,pharmaceutical compositions (e.g., pharmaceutical dosage forms) of theinvention may contain, e.g., about 0.5% to about 25% (w/v) (e.g., about0.5% to about 20% (w/v), about 0.5% to about 10% (w/v), about 0.5% toabout 2% (w/v), about 1% to about 20% (w/v), about 1% to about 10%(w/v), about 1% to about 2% (w/v), about 5% to about 20% (w/v), about 5%to about 10% (w/v), or about 7% to about 10% (w/v)) of a gelling agentrelative to solvent.

Gelling agents that may be used in the pharmaceutical compositions ofthe invention are known in the art. Non-limiting examples of gellingagents include hyaluronan, a polyoxyethylene-polyoxypropylene blockcopolymer (e.g., a poloxamer), poly(lactic-co-glycolic) acid, polylacticacid, polycaprolactone, alginic acid or a salt thereof, polyethyleneglycol, a cellulose, a cellulose ether, a carbomer (e.g., Carbopol®),agar-agar, gelatin, glucomannan, galactomannan (e.g., guar gum, locustbean gum, or tara gum), xanthan gum, chitosan, pectin, starch,tragacanth, carrageenan, polyvinylpyrrolidone, polyvinyl alcohol,paraffin, petrolatum, silicates, fibroin, and combinations thereof. Thegelling agents described herein are known in the art. Preferably, thegelling agent is hyaluronan.

A pharmaceutical composition of the invention may contain, e.g., about0.5% to about 2% (w/v) (e.g., about 1% to about 2% (w/v)) of hyaluronanrelative to solvent. A pharmaceutical composition of the invention maycontain, e.g., about 5% to about 10% (w/v) (e.g., about 6% to about 8%(w/v)) of methylcellulose relative to solvent. A pharmaceuticalcomposition of the invention may contain, e.g., hyaluronan andmethylcellulose as a gelling agent (e.g., about 0.5% to about 2% (w/v)of hyaluronan and about 5% to about 10% (w/v) of methylcelluloserelative to solvent). A pharmaceutical composition of the invention maycontain, e.g., a polyoxyethylene-polyoxypropylene block copolymer (e.g.,poloxamer) as a gelling agent. A pharmaceutical composition of theinvention may contain, e.g., about 1% to about 20% (w/v) (e.g., about 1%to about 15% (w/v), about 1% to about 10% (w/v), about 5% to about 20%(w/v), about 5% to about 15% (w/v), about 5% to about 10% (w/v), about10% to about 20% (w/v), or about 10% to about 15% (w/v)) of apolyoxyethylene-polyoxypropylene block copolymer (e.g., poloxamer)relative to solvent. The poloxamer may be poloxamer 407, poloxamer 188,or a combination thereof. A pharmaceutical composition of the inventionmay contain, e.g., about 0.5% (w/v) to about 20% (w/v) of fibroin as agelling agent relative to solvent.

Hyaluronan is a hyaluronic acid or a salt thereof (e.g., sodiumhyaluronate). Hyaluronans are known in the art and are typicallyisolated from various bacteria (e.g., Streptococcus zooepidemicus,Streptococcus equi, or Streptococcus pyrogenes) or other sources, e.g.,bovine vitreous humor or rooster combs. The weight-averaged molecularweight (Mw) of hyaluronan is typically about 50 kDa to about 10 MDa.Preferably, Mw of a hyaluronan (e.g., sodium hyaluronate) is about 500kDa to 6 MDa (e.g., about 500 kDa to about 750 kDa, about 600 kDa toabout 1.1 MDa, about 750 kDa to about 1 MDa, about 1 MDa to about 1.25MDa, about 1.25 to about 1.5 MDa, about 1.5 MDa to about 1.75 MDa, about1.75 MDa to about 2 MDa, about 2 MDa to about 2.2 MDa, about 2 MDa toabout 2.4 MDa). More preferably, the Mw of a hyaluronan (e.g., sodiumhyaluronate) is about 620 kDa to about 1.2 MDa or about 1.2 MDa to about1.9 MDa. Other preferred molecular weight ranges for a hyaluronaninclude, e.g., about 600 kDa to about 1.2 MDa.

Polyoxyethylene-polyoxypropylene block copolymers are known in the art.A non-limiting example of polyoxyethylene-polyoxypropylene blockcopolymers is a poloxamer, in which a single polyoxypropylene block isflanked by two polyoxyethylene blocks. Poloxamers are commerciallyavailable under various trade names, e.g., Synperonic®, Pluronic®,Kolliphor®, and Lutrol®. A pharmaceutical composition of the inventionmay contain, e.g., a polyoxyethylene-polyoxypropylene block copolymer(e.g., a poloxamer) includes a polyoxypropylene block with a numberaverage molecular weight (M_(n)) of, e.g., about 1,100 g/mol to about17,400 g/mol (e.g., about 2,090 g/mol to about 2,360 g/mol, about 7,680g/mol to about 9,510 g/mol, 6,830 g/mol to about 8,830 g/mol, about9,840 g/mol to about 14,600 g/mol, or about 12,700 g/mol to about 17,400g/mol). A polyoxyethylene-polyoxypropylene block copolymer (e.g., apoloxamer) may include a polyoxypropylene block with a number averagemolecular weight (Mr) of about 1,100 g/mol to about 4,000 g/mol and acalculated polyoxyethylene content of about 30% to about 85% (w/w).Preferably, a polyoxyethylene-polyoxypropylene block copolymer (e.g., apoloxamer) may include a polyoxypropylene block with a calculatedmolecular weight of, e.g., about 1,800 g/mol to about 4,000 g/mol.Preferably, the calculated polyoxyethylene content apolyoxyethylene-polyoxypropylene block copolymer (e.g., a poloxamer) maybe, e.g., about 70% to about 80% (w/w). Preferably, apolyoxyethylene-polyoxypropylene block copolymer (e.g., a poloxamer) mayhave a number average molecular weight of, e.g., about 7,680 g/mol toabout 14,600 g/mol. Non-limiting examples of poloxamers are poloxamer407 and poloxamer 188.

Celluloses and cellulose ethers are known in the art. Celluloses andcellulose ethers are commercially available under various tradenames,e.g., Avicel®, Methocel™, Natrosol®, and Tylose®. Non-limiting examplesof cellulose ethers include methylcellulose, carboxymethylcellulose,ethylcellulose, hydroxyethylcellulose, methyl hydroxyethylcellulose,hydroxypropyl methylcellulose, or hydroxypropylcellulose. A celluloseether (e.g., methylcellulose) may have a number average molecular weight(Mr) of, e.g., about 5 kDa to about 300 kDa. Methyl-substitutedcelluloses (e.g., methylcellulose, hydroxypropyl methyl cellulose, ormethyl hydroxyethylcellulose) may have methyl content of, e.g., 19% to35% (e.g., 19% to 30%).

Fibroin is a protein present in silk created by numerous insects.Fibroins are known in the art and are commercially available fromvarious vendors, e.g., Jiangsu SOHO International Group; Simatech,Suzhou, China; Xi'an Lyphar Biotech, Ltd.; Xi'an RongshengBiotechnology; Mulberry Farms, Treenway Silks, Sharda Group, ManiarEnterprises, and Wild Fibres. The molecular weight of silk fibroin istypically about 10 kDa to about 500 kDa. Fibroins are described in WO2017/139684, the disclosure of which is incorporated herein byreference.

Cross-Linked Gelling Agents

Pharmaceutical compositions of the invention may containnon-cross-linked or cross-linked gelling agents. Gelling agents may becross-linked using cross-linking agents known in the art. Preferably,the cross-linked gelling agent is covalently crosslinked. Pharmaceuticalcompositions (e.g., pharmaceutical dosage forms) including cross-linkedgelling agents may be used to control the release profile of ananti-platinum chemoprotectant agent. For example, the release of ananti-platinum chemoprotectant agent from a pharmaceutical composition(e.g., a pharmaceutical dosage form) containing a cross-linked gellingagent may be extended release relative to a reference composition thatdiffers from the pharmaceutical composition only by the lack ofcross-linking in the gelling agent in the reference composition. Theextension of the release of an anti-platinum chemoprotectant agent maybe assessed by comparing T_(max) values for the pharmaceuticalcomposition and the reference composition.

Certain gelling agents, e.g., those having carboxylate moieties (e.g.,hyaluronan, alginic acid, and carboxymethylcellulose), can becross-linked ionically using ionic cross-linking agents (e.g., amultivalent metal ion, e.g., Mg²⁺, Ca²⁺, or Al³⁺). Techniques for ioniccross-linking of gelling agents are known in the art (see, e.g., U.S.Pat. Nos. 6,497,902 and 7,790,699, the disclosures of which areincorporated herein by reference). Typically, gelling agents can beionically cross-linked in an aqueous solution using multivalent metalions, e.g., Mg²⁺, Ca²⁺, or Al³⁺, as ionic cross-linking agents. Withoutwishing to be bound by theory, the metal ions are believed to coordinateto different molecules of the gelling agent (e.g., to pendantcarboxylates residing on different molecules of the gelling agent),thereby forming a linkage between these different molecules of thegelling agent.

Certain gelling agents having reactive functional groups, e.g., —OH,—COOH, or —NH₂, may be covalently cross-linked. Techniques for covalentcross-linking of gelling agents are known in the art (see, e.g.,Khunmanee et al., J. Tissue Eng., 8: 2041731417726464, 2017, thedisclosure of which is incorporated herein by reference). Non-limitingexamples of covalent cross-linking agents include: 1,4-butanedioldiglycidyl ether (BDDE), divinyl sulfone, glutaraldehyde, cyanogenbromide, octylsuccinic anhydride, acid chlorides, diisocyanates,methacrylic anhydride, boric acid, and sodium periodate/adipic aciddihydrazide.

Other Excipients

Pharmaceutical compositions of the invention may containpharmaceutically excipients other than gelling agents. For example,pharmaceutical compositions of the invention may contain, e.g., liquidsolvents, tonicity agents, buffering agents, and/or coloring agents.Certain excipients may perform multiple roles. For example, a liquidsolvent in addition to its function as a carrier may be used as atonicity agent and/or buffering agent. Such solvents are known in theart, e.g., salines (e.g., hypertonic saline, hypotonic saline, isotonicsaline, or phosphate-buffered saline) and artificial perilymph.

Liquid solvents may be used in pharmaceutical compositions (e.g.,pharmaceutical dosage forms) of the invention as a vehicle. Liquidsolvents are known in the art. Non-limiting examples of liquid solventsinclude water, salines (e.g., hypertonic saline, hypotonic saline,isotonic saline, or phosphate-buffered saline), artificial perilymph,and tris buffer. Artificial perilymph is an aqueous solution containingNaCl (120-130 mM), KCl (3.5 mM), CaCl₂) (1.3-1.5 mM), MgCl₂ (1.2 mM),glucose (5.0-11 mM), and buffering agents (e.g., NaHCO₃ (25 mM) andNaH₂PO₄ (0.75 mM), or HEPES (20 mM) and NaOH (adjusted to pH of about7.5)).

Tonicity agents may be included in pharmaceutical compositions (e.g.,pharmaceutical dosage forms) of the invention to increase osmolarityrelative to that which is afforded by an anti-platinum chemoprotectantagent. Tonicity agents are known in the art. Non-limiting examples oftonicity agents include substantially neutral buffering agents (e.g.,phosphate buffered saline, tris buffer, or artificial perilymph),dextrose, mannitol, glycerin, potassium chloride, and sodium chloride(e.g., as a hypertonic, isotonic, or hypotonic saline). Pharmaceuticalcompositions (e.g., pharmaceutical dosage forms) of the inventioninclude sufficient amount of tonicity agents to provide foradministration to a subject a hypertonic pharmaceutical dosage form(e.g., a pharmaceutical dosage form having a calculated osmolarity of atleast 400 mOsm/L (e.g., at least 500 mOsm/L, at least 600 mOsm/L, or atleast 700 mOsm/L), and/or 2,500 mOsm/L or less (e.g., 2,000 mOsm/L,1,900 mOsm/L or less, 1,800 mOsm/L or less, 1,700 mOsm/L or less, 1,600mOsm/L or less, or 1,500 mOsm/L or less)). For example, the targetedconcentration of a tonicity agent in a pharmaceutical composition (e.g.,pharmaceutical dosage form) of the invention can be determined, e.g., by(i) subtracting the calculated osmolarity contributions of ananti-platinum chemoprotectant agent and other non-polymeric excipientsfrom the total targeted calculated osmolarity to obtain the targetedcalculated osmolarity contribution from the tonicity agent, and (ii)determining the concentration of the tonicity agent by dividing thetargeted calculated osmolarity contribution from the tonicity agent bythe number of ions and/or molecules produced upon dissolution of thetonicity agent in a liquid solvent. An appropriate amount of thetonicity agent thus can be included in the pharmaceutical composition(e.g., pharmaceutical dosage form) of the invention.

Buffering agents may be used to adjust the pH of a pharmaceuticalcomposition (e.g., a pharmaceutical dosage form) of the invention asubstantially neutral pH level. Buffering agents are known in the art.Non-limiting examples of buffering agents include, e.g., phosphatebuffers and Good's buffers (e.g., tris, MES, MOPS, TES, HEPES, HEPPS,tricine, and bicine). In addition to the pH control, buffering agentsmay be used to control osmolarity of the pharmaceutical composition(e.g., pharmaceutical dosage form) of the invention.

Methods of Use

Pharmaceutical compositions (e.g., pharmaceutical dosage forms) of theinvention may exhibit otoprotective properties against platinum-basedantineoplastic agents and may be used in a method of preventing ormitigating platinum-induced ototoxicity in subjects in need thereof. Themethod includes administration of a pharmaceutical composition of theinvention to a round window of the subject. The subject may beundergoing therapy with a platinum-based antineoplastic agent (e.g.,cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate,phenanthriplatin, picoplatin, or satraplatin).

A pharmaceutical composition of the invention may be administered to asubject, e.g., before or after the administration of a platinum-basedantineoplastic agent to the subject. Alternatively, a pharmaceuticalcomposition of the invention may be administered, e.g., at the same timeas the administration of a platinum-based antineoplastic agent. Apharmaceutical composition of the invention may be administered, e.g.,within 1 hour of the administration of a platinum-based antineoplasticagent (e.g., within 15 min, 30 min, or 1 hour before or after).Alternatively, a pharmaceutical composition of the invention may beadministered, e.g., within 24 hours of a platinum-based antineoplasticagent (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hoursbefore or after). A pharmaceutical composition of the invention may beadministered without coordination with the administration of aplatinum-based antineoplastic agent. Instead, for the period of timeduring which a subject is receiving chemotherapy including aplatinum-based antineoplastic agent, a pharmaceutical composition of theinvention may be administered once or twice daily, every other day,twice a week, or weekly.

In some embodiments, at least 50 μL (preferably, at least 100 μL; morepreferably, at least 200 μL) of the pharmaceutical composition areadministered to the round window of the subject. In particularembodiments, 1 mL or less (e.g., 0.8 mL or less or 0.5 mL or less) ofthe pharmaceutical composition are administered to the round window ofthe subject. In certain embodiments, 100 μL to 1 mL (e.g., 200 μL to 1mL, 100 μL to 0.8 mL, 200 μL to 0.8 mL, 100 μL to 0.5 mL, 200 μL to 0.5mL, 0.5 mL to 1.0 mL, 0.5 mL to 0.8 mL, or 0.8 mL to 1.0 mL) of thepharmaceutical composition are administered to the round window of thesubject.

Typically, the pharmaceutical composition of the invention may beadministered by a route different from the platinum-based antineoplasticagent. The methods of the invention may utilize a local route ofadministration, for example, the pharmaceutical composition of theinvention may be administered intratympanically or transtympanically.Transtympanic administration may include injection or infusion of aneffective amount of the pharmaceutical composition of the inventionthrough the tympanic membrane into the tympanic cavity, therebyproviding the anti-platinum chemoprotectant agent to the round window.

Methods of Preparation

A pharmaceutical composition (e.g., a pharmaceutical dosage form) of theinvention may be prepared from an anti-platinum chemoprotectant agent, agelling agent, and a liquid solvent. A method of preparing apharmaceutical composition (e.g., a pharmaceutical dosage form) of theinvention includes (i) providing the anti-platinum chemoprotectant agentand the gelling agent, and (ii) mixing the anti-platinum chemoprotectantagent and the gelling agent with the liquid solvent to produce thepharmaceutical composition.

The anti-platinum chemoprotectant agent and the gelling agent may beprovided, e.g., as a mixture or as separate ingredients. When theanti-platinum chemoprotectant agent and the gelling agent are providedseparately, the step (ii) may include, e.g.:

-   -   (a) mixing the liquid solvent first with the gelling agent to        produce an intermediate mixture and thereafter mixing the        intermediate mixture with the anti-platinum chemoprotectant        agent;    -   (b) mixing the liquid solvent first with the anti-platinum        chemoprotectant agent to produce an intermediate mixture and        thereafter mixing the intermediate mixture with the gelling        agent; or    -   (c) mixing a portion of the liquid solvent with the        anti-platinum chemoprotectant agent to produce a first mixture,        mixing another portion of the liquid solvent with the gelling        agent to produce a second mixture, and combining the first and        second mixtures.

The following examples are meant to illustrate the invention. They arenot meant to limit the invention in any way.

Examples Example 1. Formulations

Poloxamer 407 gel 1 (0.1M STS, 20% (w/v) poloxamer 407) Sodiumthiosulfate pentahydrate (106.07 mg) was dissolved in sterile, distilledwater (4.274 mL) in a sterile vial to produce a clear solution.Poloxamer 407 (855 mg; purified, non-ionic, Sigma-Aldrich) was addedinto the solution, and the resulting mixture was stirred for 15-20 minat 4° C. Evans blue (4.27 mg) was added into the vial and stirred for 10mins at 4° C. (ice/water bath).

Poloxamer 407 Gel 2 (0.5M STS, 16% (w/v) Poloxamer 407)

Poloxamer 407 gel 2 was prepared according to the procedure describedfor Poloxamer 407 gel 1 with the exception that the amount of sodiumthiosulfate pentahydrate was adjusted to provide a 0.5M concentration ofsodium thiosulfate, and the amount of poloxamer 407 was adjusted toprovide a 16% (w/v) concentration of poloxamer 407.

Preparation of Poloxamer 407 gels with 0.6M-0.8M STS, 16% (w/v)poloxamer 407 led to the observation of precipitation without gelformation.

Hyaluronan Gel 1 (0.5M STS, 1% (w/v) Hyaluronan)

Sodium thiosulfate pentahydrate (619.75 mg) was dissolved in sterile,distilled water (5 mL) in a sterile vial to produce a clear solution.Hyaluronan (50.30 mg; Pharma Grade 80, Kikkoman Biochemifa company;0.6-1.2 mDa) was added to the solution, and the resulting mixture wasstirred for 8-10 min at 4° C. The resulting solution was filteredthrough 0.22 μm Millex-GV sterile filter.

Hyaluronan Gel 2 (0.1M STS, 2% (w/v) Hyaluronan)

Sodium thiosulfate pentahydrate (124.87 mg) was dissolved in sterile,distilled water (3.031 mL). Methylcellulose (351.01 mg; Methocel® A15Premium LV, Dow Chemical Company) was dissolved in sterile, distilledwater (2.0 mL), and the resulting solution was mixed with the sodiumthiosulfate solution. Hyaluronan (100.10 mg; Pharma Grade 80, KikkomanBiochemifa company; 0.6-1.2 mDa) was added to the resulting mixture andmixed at 4° C. for 10-15 min.

Hyaluronan Gel 3 (0.5M STS, 2% (w/v) Hyaluronan)

Sodium thiosulfate pentahydrate (620.35 mg) was dissolved in sterile,distilled water (3 mL). Methylcellulose (350.23 mg; Methocel® A15Premium LV, Dow Chemical Company) was dissolved in sterile, distilledwater (2.0 mL), and the resulting solution was mixed with the sodiumthiosulfate solution. Hyaluronan (100.65 mg; Pharma Grade 80, KikkomanBiochemifa company; 0.6-1.2 mDa) was added to the resulting mixture andmixed at 4° C. for 10-15 min.

Hyaluronan Gel 4 (0.1M STS, 1% (w/v) Hyaluronan, Manitol)

Hyaluronan (50.09 mg; Pharma Grade 80, Kikkoman Biochemifa company;0.6-1.2 mDa) was added to water (5 mL). Sodium thiosulfate pentahydrate(124.9 mgs) was added. The pH of the resulting mixture was adjusted topH7.12 by addition of sodium hydroxide (1N, ca. 0.5 μL). Add appropriateamount of mannitol into the vial to adjust the osmolarity to 1.046Osm/kg. The viscous solution was filtered through 0.22 μm Millex-GVfilter.

Hyaluronan Gel 5 (0.1M STS, 1% (w/v) Hyaluronan)

Hyaluronan Gel 5 was prepared according to the procedure described forHyaluronan Gel 1 with the exception that the amount of sodiumthiosulfate pentahydrate was adjusted to provide a 0.1M concentration ofsodium thiosulfate.

Hyaluronan Gel 6 (0.2M STS, 1% (w/v) Hyaluronan)

Hyaluronan Gel 6 was prepared according to the procedure described forHyaluronan Gel 1 with the exception that the amount of sodiumthiosulfate pentahydrate was adjusted to provide a 0.2M concentration ofsodium thiosulfate.

Hyaluronan Gel 7 (0.3M STS, 1% (w/v) Hyaluronan)

Hyaluronan Gel 7 was prepared according to the procedure described forHyaluronan Gel 1 with the exception that the amount of sodiumthiosulfate pentahydrate was adjusted to provide a 0.3M concentration ofsodium thiosulfate.

Hyaluronan Gel 8 (0.4M STS, 1% (w/v) Hyaluronan)

Hyaluronan Gel 8 was prepared according to the procedure described forHyaluronan Gel 1 with the exception that the amount of sodiumthiosulfate pentahydrate was adjusted to provide a 0.4M concentration ofsodium thiosulfate.

Hyaluronan Gel 9 (0.5M STS, 1% (w/v) Hyaluronan, Tris (5×))

Hyaluronan (79.99 mg; Pharma Grade 80, Kikkoman Biochemifa company;0.6-1.2 mDa) was added to Tris buffer (8 mL, AMRESCO-0497-500G). The pHof the resulting mixture was adjusted to pH7.13 by addition of HCl (5N).Sodium thiosulfate pentahydrate (992.60 mg) was added to the abovesolution. The viscous solution was filtered through 0.22 μm Millex-GVfilter.

Hyaluronan Gel 10 (0.5M STS, 1% (w/v) Hyaluronan, Phosphate BufferedSaline (5×))

Hyaluronan (70.38 mg; Pharma Grade 80, Kikkoman Biochemifa company;0.6-1.2 mDa) was added to PBS buffer (7 mL, 5×). Sodium thiosulfatepentahydrate (868.46 mg) was added. The pH of the resulting mixture wasadjusted to pH6.99 by addition of NaOH (1N). The viscous solution wasfiltered through 0.22 μM Millex-GV filter.

Hyaluronan Gel 11 (0.8M STS, 1% (w/v) Hyaluronan)

Hyaluronan Gel 11 was prepared according to the procedure described forHyaluronan Gel 1 with the exception that the amount of sodiumthiosulfate pentahydrate was adjusted to provide a 0.8M concentration ofsodium thiosulfate.

Hyaluronan Gel 12 (1M STS, 0.8% (w/v) Hyaluronan)

Hyaluronan Gel 12 was prepared according to the procedure described forHyaluronan Gel 1 with the exception that the amount of sodiumthiosulfate pentahydrate was adjusted to provide a 1M concentration ofsodium thiosulfate, and the amount of hyaluronan was adjusted to providea 0.8% (w/v) concentration of hyaluronan.

Hyaluronan Gel 13 (0.5M STS, 0.82% (w/v) Hyaluronan (HYALGAN))

Hyaluronan Gel 13 was prepared by mixing of sodium thiosulfatepentahydrate with hyaluronan (HYALGAN, Fidia Pharma USA, Florham Park,N.J.) to afford the final preparation with 0.82% (w/v) concentration ofhyaluronan.

Hyaluronan Gel 14 (0.5M STS, 1% (w/v) Hyaluronan (SINGCLEAN))

Hyaluronan Gel 14 was prepared according to the procedure described forHyaluronan Gel 13 with the exception that hyaluronan (SINGCLEAN,Hangzhouh Singclean Medical Products Co., Ltd., Hangzhou, China) wasused in the preparation of this gel.

Hyaluronan Gel 15 (0.5M STS, 1% (w/v) Hyaluronan (EUFLEXXA))

Hyaluronan Gel 15 was prepared according to the procedure described forHyaluronan Gel 13 with the exception that hyaluronan (EUFLEXXA, FerringPharmaceuticals Inc., Parsippany, N.J.) was used in the preparation ofthis gel.

Hyaluronan Gel 16 (0.5M STS, 1% (w/v) Hyaluronan (HEALON))

Hyaluronan Gel 16 was prepared according to the procedure described forHyaluronan Gel 13 with the exception that hyaluronan (HEALON, Johnson &Johnson, New Brunswick, N.J.) was used in the preparation of this gel.

Hyaluronan Gel 17 (1M STS, 1% (w/v) Hyaluronan)

Hyaluronan Gel 17 was prepared according to the procedure described forHyaluronan Gel 1 with the exception that the amount of sodiumthiosulfate pentahydrate was adjusted to provide a 1M concentration ofsodium thiosulfate.

Hyaluronan Gel 18 (10% (w/v) N-Acetyl-L-Cysteine, 1% (w/v) Hyaluronan)

Hyaluronan (39.38 mg; Pharma Grade 80, Kikkoman Biochemifa company;0.6-1.2 mDa) was added to water (4 mL). N-Acetyl-L-cysteine (399.14 mg)was added. The pH of the resulting mixture was adjusted to pH 7.21 byaddition of NaOH (10N, 240 μL). The viscous solution was filteredthrough 0.22 μM Millex-GV filter. The osmotic pressure was measured as1.107 Osm/kg.

Other hyaluronan gels may be prepared using the procedures describedherein. For example, 1M and 1.5M hyaluronan gels may be preparedaccording to the same procedure as described for, e.g., Hyaluronan Gel 1and Hyaluronan Gel 12. Additionally, pH levels of the gels may beadjusted to pH 6.5 to 8.5 using Brønsted acids (e.g., hydrochloric acid)and bases (e.g., sodium hydroxide).

Example 2. Pharmacokinetics

Guinea Pigs, Study 1

Albino guinea pigs (Hartley), body weight at 250-350 g, were used forthe studies. For dosing, the animal was placed on its shoulder with thesurgery ear up and auditory bulla was first exposed using retroauricularapproach. A hole of 2-3 mm in diameter was drilled on the bulla toprovide direct visualization of the round window niche. Then, 10 μL ofan aqueous composition of 0.5M sodium thiosulfate/2% (w/v) hyaluronan(STS Composition) were applied onto the RWM using a 10 μL Hamiltonsyringe and a 26-gauge needle. After application, guinea pigs remainedat this position for 30 min to allow compound to diffuse into thecochlea. The bulla opening was sealed with a muscle graft and theincision closed with sutures.

Sampling procedures are as follows, in brief. All sampling proceduresare terminal. Animals were euthanized with CO₂. 0.5 mL samples of bloodwere collected by cardiac puncture. Plasma was separated bycentrifugation at 5,000 rpm at 4° C. for 10 min and collected in aseparate tube. 50 μL of cerebrospinal fluid were collected through thecisterna magma. Perilymph was collected ex vivo to avoid contaminationfrom the cerebrospinal fluid influx via the cochlear aqueduct. Thetemporal bone was rapidly isolated, and the bulla was removed to exposethe cochlea. Any visible remaining dosed compositions were carefullyremoved with absorbent points under the surgical microscope beforeperilymph sampling. A small hole was made at the apex, and then 5-7 μLof perilymph was sampled using a pulled glass pipette. All samples werefrozen immediately on dry ice and stored in −80° C. until analysis. Theconcentrations of thiosulfate in the samples were measured using themethod disclosed in Togawa et al. Chem. Pharm. Bull., 40:3000-3004,1992, the disclosure of which is incorporated herein by reference. Theresults of this study are shown in FIGS. 1A, 1B, 2A, and 2B and in Table1.

Cynomolgus Monkey

Cynomolgus monkey was administered tolfedine (4 mg/kg) subcutaneously.After 30 minutes, the animal was anesthetized via intravenous bolus ofpropofol (5.5 mg/kg). 2-3% isoflurane inhalation was then used tomaintain the animal in anesthetized state. The animal was thenimmobilized and placed laterally in reverse Trendelenburg position toensure the access to the round window. During the surgery process, theanimal was kept on a warm blanket.

Intratympanic injection in right ear was conducted when the animalreaches the anesthetized state. 1.1 mL of epinephrinehydrochloride-saline (0.1 mg in 10 mL saline) and 0.5 mL of lidocainehydrochloride (20 mg/mL) were injected subcutaneously into the skin ofear canal posterior wall of each ear respectively as local anesthetics.An incision was then made in the post-auricular skin, and part of thetemporal bone was drilled to expose middle ear. 50 μL of the STScomposition were injected into the round window membrane using a 25 Gneedle. After dosing, the animal was left on a line with its head up toallow the dosing solution to settle into the tympanic cavity for 30mins. The same procedure was then repeated for the opposite ear.

Plasma and CSF were collected ca. 2 h after dosing the 1^(st) ear(right). Right ear cochlea perilymph sampling was conducted ca. 3 hafter the right ear dosing. The animal was euthanized by IVadministration of propofol at 11 mg/kg and then exsanguinated viafemoral artery. Animal was then placed in lateral recumbent. Apost-auricular skin incision was made and the external ear canal wasextracted to expose middle ear. Part of the temporal bone was thendrilled to expose the basal turn of the cochlea. The remaining dose inthe middle ear (if visible) was cleaned with cotton tips. A drop oftissue glue was spread at the base of the cochlea to minimize thecontamination from the dosed compositions.

Using a 0.5-1 mm round-tipped burr or sharped crochet, a hole was madeat the basal turn of cochlea. Perilymph (ca. 10 μL) was then collectedusing the capillary tube inserted into the cochlear scala tympani. Thesame procedure was repeated for the left ear cochlea perilymph samplingca. 2 h after the left ear dosing. The results of this study are shownin Table 1.

TABLE 1 Poloxamer Hyaluronan Hyaluronan Hyaluronan Hyaluronan Hyaluronan407 gel 1 Gel 2 Gel 3 Gel 1 Gel 1 Gel 1 Species Guinea Pig Guinea PigGuinea Pig Guinea Pig Guinea Pig Cynomolgus Monkey Dose 10 μL (IT) 10 μL(IT) 10 μL (IT) 10 μL (IT) 50 μL (TT) 50 μL (IT) Total Dose (mg) 0.110.11 0.56 0.56 2.8 2.8 Tmax (est.) (h) 1 1 3 1.8 1 Cmax (ng/mL) 59000104040 712800 1686200 1930000 688500 @ 2 h Terminal t(½) (h) 2.38 1.532.66 2.01 2.73 AUC last (h · ng/mL) 212523 327512 4634845 906908310498230 AUC inf (h · ng/mL) 255707 377265 4724413 10418954 10703165Plasma* (ng/mL) 2390 766 In the above table, IT is intratympanicadministration, and TT is transtympanic administration. *concentrationof thiosulfate as measured in plasma samples from the tested animals.

Guinea Pigs, Study 2

Male guinea pigs weighing 200-300 g of approximately 5-7 weeks of ageserved as subjects (N=5 per group). Prior to any procedures, animalswere anesthetized using zolazepam hydrochloride (Zoletil 50; 20 mg/kg)10 minutes before surgery via the intramuscular route. If needed, anintraoperative booster was administered intraperitoneally representing aone-tenth of the original dose.

Intratympanic Injection:

-   1. Under microscopic magnification, sharp scissors were used to    create a 0.5-1.5 cm postauricular skin incision, approximately 6-8    mm caudal to the auriculo-cephalic crease. Care was exercised to    avoid cutting deeply to preserve underlying vascular structures.-   2. Careful blunt dissection through the subcutaneous fat layer,    muscles and tissues was performed with forceps. The cleidomastoideus    muscle body was gently retracted until the shiny dome of the    tympanic bulla periosteum came into view. At the caudal aspect of    the bulla, the insertion of a deeper cervical muscle, the    sternomastoideus came into view. The facial nerve, which becomes    visible at the dorsal and rostral aspect of the bulla dome, was    preserved during the operation.-   3. A self-retaining retractor was placed prior to creating a small    hole (0.5 mm diameter) either with a drilling in the posterior part    of the bulla. The bulla bone was uncapped in a dorsal and caudal    direction using a pair of jeweler's tip forceps. The bone was    removed in a piecemeal fashion under high magnification. Care was    exercised not to puncture the stapedial artery, which lies directly    beneath the bulla cap, as bleeding from this artery may compromise    the procedure. The amount of bone removed was kept to a minimum to    prevent excessive fluid entry to the middle ear while still allowing    excellent visualization and access to the round window niche.-   4. 10 or 90 μL of a gel formulation was delivered to the round    window niche using a sterile glass Hamilton syringe with 25-26 G    blunt needle.-   5. The delivered agent was allowed to rest within the round window    niche for up to 30 min. The small hole was covered with muscular    tissue and tissue glue.-   6. The incision was closed with sutures (4-0 non-absorbable    monofilament or 5-0 non-absorbable nylon) and tissue glue or wound    clips. The entire procedure took approximately 3-5 minutes depending    on agent specifications.-   7. During the procedure and until recovery, animals were placed on a    temperature controlled (38° C.) heating pad until consciousness was    regained, at which time they were returned to the home-cage.

Alternatively, the animals were administered the gel formulationstranstympanically.

Sampling Collection:

Blood Collection:

-   1. Without preinflating in the euthanasia box, the guinea pig was    placed in a box, and 100% carbon dioxide was introduced to cause the    animal unconsciousness and to reduce animal suffering. Carbon    dioxide flow was maintained for a minimum of 1 minute after the    breath has stopped. The guinea pig was removed from the euthanasia    box after death was confirmed.-   2. Blood was collected immediately after euthanasia.-   3. After the operator fixed the animal's back position, the needle    was inserted at the front of the sternal ridge at 4-6 or slightly    forward.-   4. The needle was pulled back, and the blood was returned.-   5. Volume: for each blood collection, ca. 1 mL of blood was    collected.

CSF Collection:

CSF was collected after euthanasia. A 0.5*20 intravenous infusion needlewas slowly lowered from 90° to the foramen magnum. The needle reached adistance of 4.5-5 mm under the skin, and 50-200 μl of clear tissue fluidwere withdrawn.

Perilymph Collection:

After euthanasia, the animal was stripped excess skin and muscle tissueto obtain a complete auditory bulla, and the bulla wall was cut withsmall forceps to expose the cochlea. The basal turn of bulla was cleanedby using small cotton ball. The cochlear bottom circle and the roundwindow were coated with bio glue. After drying, a unique microhole washand-drilled in the top circle of the cochlea. A 2 μL volume ofperilymph was then collected using a microcapillary inserted into thecochlear top circle. Perilymph samples were added to a vial containing18 μL of bovine serum albumin (BSA, 1M) stored at −80° C. untilanalysis.

The results of the Guinea Pig, Study 2 are provided in Tables 2 and 3.

TABLE 2 T_(max) C_(max) terminal AUC_(INF) AUC_(last) Osmol. FormulationRoute (h) (ng/mL) T_(1/2) (h) (h × ng/g) (h × ng/g) (Osm/kg) HyaluronanGel 1 TT (50 μL) 1 872360 2.73 4837831 4745200 Hyaluronan Gel 4 TT (50μL) 3 115300 2.3   608881 601694 1.046 Hyaluronan Gel 5 TT (50 μL) 1391432 6.42 4084991 3529898 0.267 Hyaluronan Gel 6 TT (50 μL) 1 10697948.89 5727631 4150041 Hyaluronan Gel 6* TT (50 μL) 3 500900 N/A N/A3817583 0.491 Hyaluronan Gel 7 TT (50 μL) 7 210933 N/A 2357105 0.657Hyaluronan Gel 7* TT (50 μL) 3 600420 N/A N/A 3281800 0.66 HyaluronanGel 8 TT (50 μL) 1 508500 3.48 4014647 3887550 0.838 Hyaluronan Gel 9 TT(50 μL) 1 1602936 2.11 7029505 6990442 2.048 Hyaluronan Gel 10 TT (50μL) 1 1293624 2.2  5866461 5825930 Hyaluronan Gel 11 TT (50 μL) 11371800 2.99 5863793 5714356 1.494 Hyaluronan Gel 12 TT (50 μL) 11892000 2.57 7744025 7644025 1.860 Hyaluronan Gel 17 TT (50 μL) 11044400 2.47 5918627 5843593 Hyaluronan Gel 18 TT (50 μL) 1 705600 5.534887980 4876589 1.107 In this table, TT is transtympanic administration,*This test was a duplicate of the preceding test.

TABLE 3 MW T_(max) C_(max) terminal AUC_(INF) AUC_(last) Formulation(mDa) Route (h) (ng/mL) T_(1/2) (h × ng/g) (h × ng/g) Hyaluronan Gel 10.6-1.2  IT (10 μL) 1 625297 1.88 4112197 4099226 Hyaluronan Gel 13 0.5-0.73 TT (50 μL) 1 815408 2.82 4868652 4754315 Hyaluronan Gel 14unknown TT (50 μL) 1 851568 3.27 5178077 4992602 Hyaluronan Gel 152.4-3.6 TT (50 μL) 1 628200 3.41 3908302 3761700 Hyaluronan Gel 16 ca. 4TT (50 μL) 1 919097 5.42 5033151 4407366 In this table, IT isintratympanic administration, and TT is transtympanic administration.

Example 3. Pharmacodynamics

Cisplatin was diluted with 0.9% (w/v) saline to a final concentration of5 mg/mL. Albino guinea pigs (Hartley), body weight at 250-350 g wereused in the study. After a minimal 3 days acclamation, 28 animals wereenrolled into the study. Under aseptic condition, cisplatin wasadministered intraperitoneally with a bolus injection. The five cohortswere staggered with different starting dates for the study.

Seven days after the cisplatin administration, the animals were recordedfor their auditory brainstem responses (ABR) response using TDT RZ6Multi-I/O processor. Historical ABR data were used to define a baseline.The animals were anesthetized with tiletamine hydrochloride andzolazepam hydrochloride (Zoletil). Acoustic stimuli were delivered viaan earphone. Needle electrodes were placed near the ear canal at thecausoventral position, the vertex of the skull, and a ground at thelower leg. The stimulus level was from 10 to 90 dB in 5 dB steps, andthe tone-pip frequencies were 4, 24, and 32 kHz. The ceiling soundpressure level was 90 dB. ABR threshold was observed by visualinspection of stacked waveforms as the lowest sound pressure level, atwhich the waveform was above the noise floor.

Prior to the cisplatin study, ABR data from 50 animals were recorded forboth ears of each animal (Naïve n=100). The threshold at 32 kHz in naïveanimals was 39.8 dB. The range of normal hearing was defined as amean±2SD, 27.9 to 51.6 dB. Cisplatin primarily induces hearing loss athigh frequencies. A clear pattern of hearing loss after cisplatin isdefined as a threshold of 60 dB and above at 32 kHz.

In this study, 1 out of 28 animals died before the day 7 measurement. Inthe remaining 27 animals, 18 animals had hearing loss with threshold >60dB at 32 kHz (FIG. 3A). The range of hearing loss at 32 kHz was thethresholds from 65 dB to 90 dB (FIG. 3B). 90 dB is the measurementceiling. Note when no waveform or waveform only seen at 90 dB, thethreshold was defined both as 90 dB. The average threshold at 32 kHz was82 dB, which corresponds to an average 42.2 dB shift from the naïvethreshold of 39.8 dB (FIG. 4A).

Local Intratympanic Dosing and Cochlear Sampling

Local intratympanic dosing and cochlear sampling were conducted asdescribed in Example 2.

Locally Delivered Anti-Platinum Chemoprotectant Agent Provides HearingProtection from Platinum-Based Antineoplastic Agent

Evaluation of the effect of a locally delivered anti-platinumchemoprotectant agent on hearing protection from a platinum-basedantineoplastic agent was conducted as follows.

An aqueous composition of 0.5M sodium thiosulfate/2% (w/v) hyaluronan(STS Composition) or vehicle was dosed intratympanically onto the roundwindow in the left ear (LE) as described above, and the right ear (RE)was left untreated in the guinea pigs (FIG. 5). 60 min after STSComposition or vehicle dosing, the animals were injected with cisplatinat 10 mg/kg intraperitoneally. ABR at 4, 24, and 32 kHz was measured inboth ears 7 days after cisplatin administration.

Because of heterogeneity of hearing loss after cisplatin challenge, theuntreated right ears was used to select the animals with hearing loss.There were 21 animals with right ear threshold >60 dB at 32 kHz. Ofthese 21 animals, 3 with otitis media were excluded, leaving 18 animalsfor the final analysis. 10 animals were dosed with vehicle and 8 animalswere dosed with STS Composition (FIG. 4B). In the untreated right earsof both the STS Composition and vehicle groups, there is no differencein the ABR thresholds with an average threshold 73 dB at 4 kHz, 71 dB at24 kHz, and 80 dB at 32 kHz. The vehicle-treated left ears had nosignificant difference in comparison to their untreated right ears,showing thresholds 74 dB at 4 kHz, 70 dB at 24 kHz, and 74 dB at 32 kHz.

The STS Composition-treated ears had significantly lower thresholds atboth 32 kHz and 24 KHz compared to the vehicle-treated ears anduntreated right ears (***P<0.001, two way ANOVA). At 4 kHz an averagethreshold was 61 dB in the STS Composition treated ears and 75 dB intheir untreated contralateral right ears; the protection was notstatistically significant (P=0.089). The average thresholds in the STSComposition treated ears were 40 dB and 48 dB at 24 kHz and 32 kHz,respectively, in contrast to 69 dB and 80 dB in their contralateraluntreated right ears. The normal hearing thresholds were 35 dB and 40 dBat 24 kHz and at 32 kHz, respectively, in the naïve animals. To thenaïve ears, the untreated ears after cisplatin had an average of 34 dBand 40 dB threshold elevation at 24 kHz and 32 kHz, respectively, butSTS Composition treated ears only had 5 dB and 8 dB shift. Therefore,sodium thiosulfate provided, on average, 80% protection at both 24 kHzand 32 kHz.

In a similarly designed study as described hereinabove, sound pressurelevels at 4, 24, and 32 kHz were measured during ABR tests for theguinea pigs administered vehicle or sodium thiosulfate (0.1M, 0.5M, or1M sodium thiosulfate gel) to one ear each followed by a cisplatinchallenge (Cisplatin 10MPK, intravenous injection). Different doses ofhyaluronan gels were administered as a 10 μL IT injection into the leftear one hour prior to cisplatin administration. The contralateral ear(right ear) of the animal was untreated. Hyaluronan Gel 5 (0.1M),Hyaluronan Gel 1 (0.5M), and Hyaluronan Gel 17 (1M) was tested. Theuntreated ears demonstrated significant threshold shifts compared tonaïve animals (gray shaded areas). The groups treated with HyaluronanGel 1 (0.5M) and Hyaluronan Gel 17 (1M) showed hearing protectioncompared to the untreated contralateral control ears at all testedfrequencies. No protection was seen with the vehicle treated ears. Theresults are summarized in FIG. 6.

OTHER EMBODIMENTS

Various modifications and variations of the described invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific embodiments, it should be understood thatthe invention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in the artare intended to be within the scope of the invention.

Other embodiments are in the claims.

The invention claimed is:
 1. A method of preventing or mitigatingplatinum-induced ototoxicity in a subject to whom a platinum-basedanti-neoplastic agent is administered, the method comprisingadministering to the round window of the subject an effective amount ofa hypertonic pharmaceutical composition having a calculated osmolarityof 1,500-7,000 mOsm/L and comprising 0.5M-2.0M of an anti-platinumchemoprotectant agent, a gelling agent, and an optional tonicity agent;wherein the calculated osmolarity is calculated based on thechemoprotectant agent and the tonicity agent, if present, wherein thehypertonic pharmaceutical composition is administered prior toadministration of the platinum-based anti-neoplastic agent.
 2. Themethod of claim 1, wherein the effective amount of the hypertonicpharmaceutical composition is administered intratympanically ortranstympanically.
 3. The method of claim 1, wherein the hypertonicpharmaceutical composition is administered by a route different from theplatinum-based antineoplastic agent.
 4. The method of claim 1, wherein50 μL to 1 mL of the hypertonic pharmaceutical composition areadministered to the round window of the subject.
 5. The method of claim1, wherein the hypertonic pharmaceutical composition is administeredwithin 1 hour prior to administration of the platinum-basedantineoplastic agent.
 6. The method of claim 1, wherein the hypertonicpharmaceutical composition is administered within 2 hours prior toadministration of the platinum-based antineoplastic agent.
 7. The methodof claim 1, wherein the hypertonic pharmaceutical composition isadministered within 3 hours prior to administration of theplatinum-based antineoplastic agent.
 8. The method of claim 1, whereinthe hypertonic pharmaceutical composition is administered within 4 hoursprior to administration of the platinum-based antineoplastic agent. 9.The method of claim 1, wherein the hypertonic pharmaceutical compositionis administered within 5 hours prior to administration of theplatinum-based antineoplastic agent.
 10. The method of claim 1, whereinthe hypertonic pharmaceutical composition is administered within 6 hoursprior to administration of the platinum-based antineoplastic agent. 11.A method of preventing or mitigating platinum-induced ototoxicity in asubject to whom a platinum-based anti-neoplastic agent is administered,the method comprising administering to the round window of the subjectan effective amount of a hypertonic pharmaceutical composition having acalculated osmolarity of 1,500-7,000 mOsm/L and comprising 0.5M-2.0M ofan anti-platinum chemoprotectant agent, a gelling agent, and an optionaltonicity agent; wherein the calculated osmolarity is calculated based onthe chemoprotectant agent and the tonicity agent, if present, whereinthe hypertonic pharmaceutical composition is administered afteradministration of the platinum-based agent.
 12. The method of claim 11,wherein the effective amount of the hypertonic pharmaceuticalcomposition is administered intratympanically or transtympanically. 13.The method of claim 11, wherein the hypertonic pharmaceuticalcomposition is administered by a route different from the platinum-basedantineoplastic agent.
 14. The method of claim 11, wherein 50 μL to 1 mLof the hypertonic pharmaceutical composition are administered to theround window of the subject.
 15. The method of claim 11, wherein thehypertonic pharmaceutical composition is administered within 1 hourafter administration of the platinum-based antineoplastic agent.
 16. Themethod of claim 11, wherein the hypertonic pharmaceutical composition isadministered within 2 hours after administration of the platinum-basedantineoplastic agent.
 17. The method of claim 11, wherein the hypertonicpharmaceutical composition is administered within 3 hours afteradministration of the platinum-based antineoplastic agent.
 18. Themethod of claim 11, wherein the hypertonic pharmaceutical composition isadministered within 4 hours after administration of the platinum-basedantineoplastic agent.
 19. The method of claim 11, wherein the hypertonicpharmaceutical composition is administered within 5 hours afteradministration of the platinum-based antineoplastic agent.
 20. Themethod of claim 11, wherein the hypertonic pharmaceutical composition isadministered within 6 hours after administration of the platinum-basedantineoplastic agent.